This invention relates to methods for the stabilization, storage and delivery of biologically active macromolecules, such as proteins, peptides and nucleic acids. In particular, this invention relates to protein or nucleic acid crystals, formulations and compositions comprising them. Methods are provided for the crystallization of proteins and nucleic acids and for the preparation of stabilized protein or nucleic acid crystals for use in dry or slurry formulations. The crystals, crystal formulations and compositions of this invention can be reconstituted with a diluent for the parenteral administration of biologically active macromolecular components.
The methods of this invention are useful for preparing crystals of xe2x80x9cnakedxe2x80x9d DNA and RNA sequences that code for therapeutic or immunogenic proteins and can be administered parenterally. The dissolving DNA and RNA molecules, subsequently taken up by the cells and used to express the protein with the proper glycosylation pattern, can be either therapeutic or immunogenic. Alternatively, the present invention is useful for preparing crystals, crystal formulations and compositions of sense and antisense polynucleotides of RNA or DNA.
The present invention is further directed to encapsulating proteins, glycoproteins, enzymes, antibodies, hormones and peptide crystals or crystal formulations into compositions for biological delivery to humans and animals. According to this invention, protein crystals or crystal formulations are encapsulated within a matrix comprising a polymeric carrier to form a composition. The formulations and compositions enhance preservation of the native biologically active tertiary structure of the proteins and create a reservoir which can slowly release active protein where and when it is needed. Such polymeric carriers include biocompatible and biodegradable polymers. The biologically active protein is subsequently released in a controlled manner over a period of time, as determined by the particular encapsulation technique, polymer formulation, crystal geometry, crystal solubility, crystal crosslinking and formulation conditions used. Methods are provided for crystallizing proteins, preparing stabilized formulations using pharmaceutical ingredients or excipients and optionally encapsulating them in a polymeric carrier to produce compositions and using such protein crystal formulations and compositions for biomedical applications, including delivery of therapeutic proteins and vaccines. Additional uses for the protein crystal formulations and compositions of this invention involve protein delivery in human food, agricultural feeds, veterinary compositions, diagnostics, cosmetics and personal care compositions.
Proteins are used in a wide range of applications in the fields of pharmaceuticals, veterinary products, cosmetics and other consumer products, foods, feeds, diagnostics, industrial chemistry and decontamination. At times, such uses have been limited by constraints inherent in proteins themselves or imposed by the environment or media in which they are used. Such constraints may result in poor stability of the proteins, variability of performance or high cost.
It is imperative that the higher order three-dimensional architecture or tertiary structure of a protein be preserved until such time that the individual protein molecules are required to perform their unique function. To date, a limiting factor for use of proteins, particularly in therapeutic regimens, remains the sensitivity of protein structure to chemical and physical denaturation encountered during delivery.
Various approaches have been employed to overcome these barriers. However, these approaches often incur either loss of protein activity or the additional expense of protein stabilizing carriers or formulations.
One approach to overcoming barriers to the widespread use of proteins is crosslinked enzyme crystal (xe2x80x9cCLEC(trademark)xe2x80x9d) technology [N. L. St. Clair and M. A. Navia, J. Am. Chem. Soc., 114, pp. 4314-16 (1992)]. See also PCT patent application PCT/US91/05415. Crosslinked enzyme crystals retain their activity in environments that are normally incompatible with enzyme function. Such environments include prolonged exposure to proteases, organic solvents, high temperature or extremes of pH. In such environments, crosslinked enzyme crystals remain insoluble, stable and active.
Despite recent progress in protein technology generally, two problems which are discussed below continue to limit the use of biological macromolecules in industry and medicine. The first problem relates to molecular stability and sensitivity of higher order tertiary structures to chemical and physical denaturation during manufacturing and storage. Second, the field of biological delivery of therapeutic proteins requires that vehicles be provided which release native proteins, such as proteins, glycoproteins, enzymes, antibodies, hormones, nucleic acids and peptides at a rate that is consistent with the needs of the particular patient or the disease process.
Macromolecule Stability
Numerous factors differentiate biological macromolecules from conventional chemical entities, such as for example, their size, conformation and amphiphilic nature. Macromolecules are not only susceptible to chemical, but also physical degradation. They are sensitive to a variety of environmental factors, such as temperature, oxidizing agents, pH, freezing, shaking and shear stress [Cholewinski, M., Luckel, B. and Horn, H., Acta Helv., 71, 405 (1996)]. In considering a macromolecule for drug development, stability factors must be considered when choosing a production process.
Maintenance of biological activity during the development and manufacture of pharmaceutical products depends on the inherent stability of the macromolecule, as well as the stabilization techniques employed. A range of protein stabilization techniques exist; including:
a) Addition of chemical xe2x80x9cstabilizersxe2x80x9d to the aqueous solution or suspension of protein. For example, U.S. Pat. Nos. 4,297,344 discloses stabilization of coagulation factors II and VIII, antithrombin III and plasminogen against heat by adding selected amino acids. U.S. Pat. No. 4,783,441 discloses a method for stabilizing proteins by adding surface-active substances. U.S. Pat. No. 4,812,557 discloses a method for stabilizing interleukin-2 using human serum albumin. The drawback of such methods is that each formulation is specific to the protein of interest and requires significant development efforts.
b) Freeze/thaw methods in which the preparation is mixed with a cryoprotectant and stored at very low temperatures. However, not all proteins will survive a freeze/thaw cycle.
c) Cold storage with cryoprotectant additive, normally glycerol.
d) Storage in the glass form, as described in U.S. Pat. No. 5,098,893. In this case, proteins are dissolved in water-soluble or water-swellable substances which are in amorphous or glassy state.
e) The most widely used method for the stabilization of proteins is freeze-drying or lyophilization [Carpenter, J. F., Pical, M. J., Chang, B. S. and Randolph, T. W., Pharm. Res., 14:(8) 969 (1997)]. Whenever sufficient protein stability cannot be achieved in aqueous solution, lyophilization provides the most viable alternative. One disadvantage of lyophilization is that it requires sophisticated processing, is time consuming and expensive [Carpenter, J. F., Pical, M. J., Chang, B. S. and Randolph, T. W., Pharm. Res., 14:(8) 969 (1997) and literature cited therein]. In addition, if lyophilization is not carried out carefully, most preparations are at least partially denatured by the freezing and dehydration steps of the technique. The result is frequently irreversible aggregation of a portion of protein molecules, rendering a formulation unacceptable for parenteral administration.
The vast majority of protein formulations produced by the above-described techniques require cold storage, sometimes as low as xe2x88x9220xc2x0 C. Exposure to elevated temperatures during shipping or storage can result in significant activity losses. Thus, storage at elevated, or even ambient temperatures, is not possible for many proteins.
Proteins, peptides and nucleic acids are increasingly employed in the pharmaceutical, diagnostic, food, cosmetic, detergent and research industries. There is a great need for alternative stabilization procedures, which are fast, inexpensive and applicable to a broad range of biological macromolecules. In particular, stabilization procedures are needed that do not rely on the excessive use of excipients, which can interfere with the functions of those biological macromolecules.
The stability of small molecule crystalline drugs is such that they can withstand extreme forces during the formulation process (see U.S. Pat. No. 5,510,118). Forces associated with milling nanoparticles of crystalline material of relatively insoluble drugs include: shear stress, turbulent flow, high impact collisions, cavitation and grinding. Small molecular crystalline compounds have been recognized as being much more stable toward chemical degradation than the corresponding amorphous solid [Pical, M. J., Lukes, A. L., Lang, J. E. and Gaines, J. Pharm. Sci., 67, 767 (1978)]. Unfortunately, crystals of macromolecules, such as proteins and nucleic acids, present additional problems and difficulties not associated with small molecules.
For most of this century, science and medicine have tried to solve the problem of providing insulin in a useful form to diabetics. Attempts have been made to solve some of the problems of stability and biological delivery of that protein. For example, U.S. Pat. No. 5,506,203 describes the use of amorphous insulin combined with an absorbtion enhancer. The solid state insulin was exclusively amorphous material, as shown by a polarized light microscope.
Jensen et al. co-precipitated insulin with an absorbtion enhancer for use in respiratory tract delivery of insulin (See PCT patent application Wo 98/42368). Here, the absorbtion enhancer was desribed as a surfactant, such as a salt of a fatty acid or a bile salt. Insulin crystals of less than 10 micrometers in diameter and lacking zinc were produced by S. Havelund (See PCT patent application Wo 98/42749). Similarly, crystals were also produced in the presence of surfactants to enhance pulmonary administration.
To date, those of skill in the art recognize that the greatly enhanced stability of the crystalline state observed for small molecules does not translate to biological macromolecules [Pical, M. J. and Rigsbee, D. R., Pharm. Res., 14:1379 (1997)]. For example, aqueous suspensions of crystalline insulin are only slightly more stable (to the degree of a factor of two) than corresponding suspensions of amorphous phase [Brange, J., Langkjaer, L., Havelund, S. and Volund, A., Pharm. Res., 9:715 (1992)]. In the solid state, lyophilized amorphous insulin is far more stable than lyophilized crystalline insulin under all conditions investigated [Pical, M. J. and Rigsbee, D. R., Pharm. Res., 14:1379 (1997)].
Until now, formulations of crystalline proteins have been available only for very small proteins, e.g. proteins with molecular weights of less than 10,000 Daltons. Molecular weight has profound effect on all properties of macromolecules, including their macromolecular volume, hydration, viscosity, diffusion, mobility and stability. [Cantor, C. R and Schimmel, P. R, Biophysical Chemistry, W. H. Freeman and Co., New York, 1980].
We have found, surprisingly, that biological macromolecules which are not stable when held in solution at ambient or elevated temperatures can nevertheless be successfully stored in dry form for long periods of time at such temperatures in crystalline form. As a practical matter, five aspects of this discovery are particularly advantageous.
First, crystallinity of stored materials is very important, since large scale crystallization can be introduced as a final purification step and/or concentration step in clinical manufacturing processes, such as those for manufacturing therapeutics and vaccines. Moreover, large scale crystallization can replace some of the purification steps in the manufacturing process. For example, protein crystallization can streamline the production of protein formulations making it more affordable.
Second, macromolecular interactions which occur in solution are prevented or severely reduced in the crystalline state, due to considerable reduction of all reaction rates. Thus, the crystalline state is uniquely suited to the storage of mixtures of biological macromolecules.
Third, solid crystalline preparations can be easily reconstituted to generate ready to use parenteral formulations having very high protein concentration. Such protein concentrations are considered to be particularly useful where the formulation is intended for subcutaneous administration. (See PCT patent application Wo 97/04801). For subcutaneous administration, injection volumes of 1.5 ml or less are well tolerated. Thus, for proteins that are dosed at 1 mg/kg on a weekly basis a protein concentration of at least 50 mg/ml is required and 100-200 mg/ml is preferred. These concentrations are difficult to achieve in liquid formulations, due to the aggregation problems. They can easily be achieved in the crystalline formulations of this invention.
Fourth, protein crystals also constitute a particularly advantageous form for pharmaceutical dosage preparation. The crystals may be used as a basis for slow release formulations in vivo. As those of skill in the art will appreciate, particle size is of importance for the dissolution of crystals and release of activity. It is also known that the rate of release is more predictable if the crystals have substantially uniform particle size and do not contain amorphous precipitate (see European patent 0 265 214). Thus, protein crystals may be advantageously used (see PCT patent application WO 96/40049), on implantable devices. Implant reservoirs are generally on the order of 25-250 xcexcl. With this volume restriction, a formulation of high concentration (greater than 10%) and a minimum amount of suspension vehicle is preferred. Protein crystals of this invention may be easily formulated in non-aqueous suspensions in such high concentrations.
Fifth, another advantage of crystals is that certain variables can be manipulated to modulate the release of macromolecules over time. For example, crystal size, shape, formulation with excipients that effect dissolution, crosslinking, level of crosslinking and encapsulation into a polymer matrix can all be manipulated to produce delivery vehicles for biological molecules.
The present invention overcomes the above-described obstacles by employing the most stable form of an active protein, the crystalline form and either (1) adding ingredients or excipients where necessary to stabilize dried crystals or (2) encapsulating the protein crystals or crystal formulations within a polymeric carrier to produce a composition that contains each crystal and subsequently allows the release of active protein molecules. Any form of protein, including glycoproteins, antibodies, enzymes, hormones or peptides, may be crystallized and stabilized or encapsulated into compositions according to the methods of this invention. In addition, the nucleic acids coding for such proteins may be similarly treated.
The crystal(s) may be encapsulated using a variety of polymeric carriers having unique properties suitable for delivery to different and specific environments or for effecting specific functions. The rate of dissolution of the compositions and, therefore, delivery of the active protein can be modulated by varying crystal size, polymer composition, polymer crosslinking, crystal crosslinking, polymer thickness, polymer hydrophobicity, polymer crystallinity or polymer solubility.
The addition of ingredients or excipients to the crystals of the present invention or the encapsulation of protein crystals or crystal formulations results in further stabilization of the protein constituent.